A glycoside analog of mammalian oligomannose formulated with a TLR4-stimulating adjuvant elicits HIV-1 cross-reactive antibodies

Abstract

The occurrence of oligomannose-specific broadly neutralizing antibodies (bnAbs) has spurred efforts to develop immunogens that can elicit similar antibodies. Here, we report on the antigenicity and immunogenicity of a CRM₁₉₇-conjugate of a previously reported oligomannose mimetic. Oligomannose-specific bnAbs that are less dependent on interactions with the HIV envelope protein sequence showed strong binding to the glycoconjugates, with affinities approximating those reported for their cognate epitope. The glycoconjugate is also recognized by inferred germline precursors of oligomannose-specific bnAbs, albeit with the expected low avidity, supporting its potential as an immunogen. Immunization of human-antibody transgenic mice revealed that only a TLR4-stimulating adjuvant formulation resulted in antibodies able to bind a panel of recombinant HIV trimers. These antibodies bound at relatively modest levels, possibly explaining their inability to neutralize HIV infectivity. Nevertheless, these findings contribute further to understanding conditions for eliciting HIV-cross-reactive oligomannose-specific antibodies and inform on next steps for improving on the elicited response.

Figure 1

Schematic of neoglycoconjugate NIT211. Shown is the chemical structure of a synthetic oligomannose mimetic conjugated onto CRM₁₉₇. The number of glycoside molecules per CRM₁₉₇ for each conjugate was determined by MALDI-TOF and ranged from 2–6 glycosides. Image adapted from Trattnig et al.

Figure 2

NIT211 conjugate is recognized by oligomannose-specific bnAbs and their germline precursors. NIT211 (4.1 glycosides per CRM₁₉₇) was coated as solid-phase antigen onto ELISA plate wells at 5 µg/ml in PBS and assayed for recognition by the different antibodies. All antibodies were expressed recombinantly as human IgG1. (A) Binding of PGT128/130 bnAb family members PGT125, PGT126, PGT128 and PGT130. (B) Binding of non-PGT128/130 family antibodies BF520.1, BG18, PCDN-33A, PGDM12, PGDM21, PCDN76-33A, VRC41.01. (C) Binding of inferred gl precursors BF520.1, BG18, PCDN-33A and the PGT128/130 family. Results are from single experiments performed with technical duplicates.

Figure 3

Binding affinities of PGT125, PGT126, PGT128 and PGT130 Fabs for NIT211 as measured by SPR. (A) Geometric mean of the association constant (ka), dissociation constant (kd) and equilibrium dissociation constant (K_D) for the four PGT bnAbs. The results are from 2–3 independent experiments. (B) Representative sensorgrams of Fab binding to NIT211 using single-cycle kinetics. The calculcated association constant (ka), dissociation constant (kd) and K_D are indicated. The black lines show the data (grey) fitted to a 1:1 binding model.

Figure 4

The relative binding affinities of PGT antibodies for NIT211 increase with increasing ligand density. Binding to NIT211 conjugates was assessed by ELISA. NIT211 (2.6 ligands, 4.1, and 6.2 ligands) was coated as solid-phase antigen onto ELISA plate wells at 5 µg/ml (82, 79, 76 nM respectively) and assayed for antibody binding. (A) Binding of PGT125, PGT126, PGT128 and PGT130 IgG to NIT211 at three different densities of glycoside per CRM₁₉₇. (B) Binding of PGT125, PGT126, PGT128 and PGT130 Fabs to NIT211 at two different densities of glycoside per CRM₁₉₇.

Figure 5

Only animals administered GLA-SE-adjuvanted NIT211 mount an IgG response to the oligomannose mimetic that is of the IgG3 subclass. Trianni mice (n = 5 per group) were immunized subcutaneously (days 0, 21, 42 and 105) and sera collected on day 0 prior to immunization and on days 10, 28, 49, and 119 post-immunization. (A) Binding of total IgG antibodies in pre-immune and post-immune sera to BSA-conjugated oligomannose mimetic. Binding curves represent mean values for the five animals in each immunization group, each tested in duplicate, with error bars denoting the standard deviation from the mean. (B) Individual IgG binding curves for NIT211 + GLA-SE immunized mice (Ms1 to 5). The sex of each mouse is also noted. (C) IgG1, IgG2b, IgG2c and IgG3 antibody subclass responses in day 119 post-immune sera of NIT211 + GLA-SE immunized animals for the BSA-conjugated oligomannose mimetic in comparison to the CRM₁₉₇ protein carrier.

Figure 6

NIT211 formulated in GLA-SE elicits antibodies with capacity to bind recombinant HIV Env trimers. (A) Binding of total IgG from day 119 sera from unimmunized, and NIT211-immunized animal groups to SOSIP trimers AMC008 (subtype B), Du422 (subtype C), ZM197M (subtype C), BG505 (subtype A), B41 (subtype B), and CZA97 (subtype C). The HIS-tagged trimers were captured on nickel-coated ELISA plates at 5 µg/ml in PBS. (B) Level of IgG1, IgG2, IgG3 antibody subclass binding (1:100 dilution) to B41 SOSIP trimer in day 119 sera of NIT211 + GLA-SE immunized animals. (C) Residual binding of bnAb PGT128 to B41 SOSIP trimer following incubation with sera from NIT211 + GLA-SE immunized animals (day 119) vs sera from KLH + Alum/CpG ODN1826 immunized animals (day 34). (D) Day 119 sera from NIT211 + GLA-SE immunized animals and sera from a group of unimmunized animals were assessed for pseudovirus neutralization using a panel of seven diverse HIV-1 strains (92TH021, 92RW020, 94UG103, 92BR020, 97ZA012, JRCSF and NL4-3). Pseudotyped vesicular stomatitis virus (VSV) was used as a negative control. All graphs depict mean values for the serum samples from all animals (n = 5) in each group. Error bars represent standard error from the mean.